This invention relates to a process for preparing a reagent for measuring endotoxin by using an extracted solution of hemocyte lysate (amoebocyte lysate) of horseshoe crab (hereinafter abbreviated as "AL solution") as a starting material, the reagent thus prepared and processes for measuring endotoxin using said reagent.
Endotoxins (hereinafter abbreviated as "ET's") are lipopolysaccharides present mainly in cell wall of Gram-negative bacteria and are known as pyrogens. Therefore, the measurement of ET concentration in a sample is one important measurement in the fields of medical science, pharmacy and microbiology.
At present, as a method for measuring ET, the so-called Limulus test utilizing the phenomenon that AL solution is activated by ET to form gel clot is widely employed because of its simplicity, convenience, low cost, etc.
However, it was found that AL solution reacts not only with ET's but also with carboxymethylated .beta.-1,3-glucan [Kakinuma et al., Biochem. Biophys. Research Communication, 101(2), 434-439 (1981)]. It was proved that this phenomenon is caused by the reaction of a factor (hereinafter abbreviated as "GL-sensitive factor") present in AL solution which reacts with .beta.-1,3-glucan (hereinafter abbreviated as "GL") to trigger coagulation with GL or a derivative thereof (Bacterial Endotoxin, published by Verlay Chemic, 365-382, 1984).
Therefore, most of commercially available Limulus test reagents react not only with ET but also with GL, so that it is difficult to judge which of ET, GL and a mixture thereof is present in a sample, by the Limulus test. Thus, the specificity of such Limulus test reagents is a problem.
In order to solve this problem, there has been reported a method for preparing a reagent specific for ET by removing GL-sensitive factor from AL solution [Japanese Patent Appln. Kokai (Laid-Open) Nos. 58-13516 and 59-27828]. However, all the methods disclosed in these references require a very troublesome procedure of treating AL solution, for example, by a gel filtration method or a chromatographic method using a carrier having heparin or dextran sulfate attached thereto, to separate the AL solution into a fraction of proclatting enzyme, a fraction of GL-sensitive factor, and a fraction of a factor (hereinafter abbreviated as "ET-sensitive factor") which reacts with ET to trigger coagulation in order to remove the GL-sensitive factor. Therefore, for preventing AL solution or the fractions obtained therefrom from being contaminated by ET during the separation procedures, there are required, for example, facilities used exclusively for carrying out said procedures. Moreover, the above methods are further disadvantageous in that the individual fractions should be properly mixed again in order to obtain a reagent specific for ET.